Chimeric antigen receptor (CAR) comprising a CD19-binding domain

ABSTRACT

There is provided a chimeric antigen receptor (CAR) comprising a CD19-binding domain which comprises a) a heavy chain variable region (VH) having complementarity determining regions (CDRs) with the following sequences: CDR1—GY-AFSSS (SEQ ID No. 1); CDR2—YPGDED (SEQ ID No. 2) CDR3—SLLYGDYLDY (SEQ ID No. 3); and b) a light chain variable region (VL) having CDRs with the following sequences: CDR1—SASSSVSYMH (SEQ ID No. 4); CDR2—DTSKLAS (SEQ ID No. 5) CDR3—QQWNINPLT (SEQ ID No. 6). There is also provided a cell comprising such a CAR, and the use of such a cell in the treatment of cancer, in particular a B cell malignancy.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is that National Stage of International Application No.PCT/GB2016/050574, filed on Mar. 4, 2016, and claims the benefit ofpriority to GB Application No. 1503742.7, filed on Mar. 5, 2015, both ofwhich are hereby incorporated by referenced in their entireties for allpurposes.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy was created on Aug. 30, 2017,is named DYC_011_US1_SL.txt and is 66,276 bytes in size.

FIELD OF THE INVENTION

The present invention relates to a chimeric antigen receptor (CAR) whichbinds the B-lymphocyte antigen CD19 (Cluster of Differentiation 19). Tcells expressing such a CAR are useful in the treatment of cancerousdiseases such as B-cell leukemias and lymphomas.

BACKGROUND TO THE INVENTION

Chimeric Antigen Receptors

Traditionally, antigen-specific T-cells have been generated by selectiveexpansion of peripheral blood T-cells natively specific for the targetantigen. However, it is difficult and quite often impossible to selectand expand large numbers of T-cells specific for most cancer antigens.Gene-therapy with integrating vectors affords us a solution to thisproblem: transgenic expression of Chimeric Antigen Receptor (CAR) allowslarge numbers of T-cells specific to any surface antigen to be easilygenerated by ex vivo viral vector transduction of a bulk population ofperipheral blood T-cells.

The most common forms of these molecules are fusions of single-chainvariable fragments (scFv) derived from monoclonal antibodies whichrecognise a target antigen, fused via a spacer and a transmembranedomain to a signalling endodomain. Such molecules result in activationof the T-cell in response to recognition by the scFv of its cognatetarget. When T cells express such a CAR, they recognize and kill targetcells that express the target antigen. Several CARs have been developedagainst tumour associated antigens, and adoptive transfer approachesusing such CAR-expressing T cells are currently in clinical trial forthe treatment of various cancers. To-date however, the main clinicalexploration and potential application of CAR therapy is as treatment forB-cell malignancies.

CARs Directed Against CD19

CD19 is a B-cell antigen which is expressed very early in B-celldifferentiation and is only lost at terminal B-cell differentiation intoplasma cells. Hence, CD19 is expressed on all B-cell malignancies apartfrom multiple myeloma. It is not expressed on other haematopoieticpopulations or non-haematopoietic cells and therefore targeting thisantigen should not lead to toxicity to the bone marrow ornon-haematopoietic organs. Loss of the normal B-cell compartment isconsidered an acceptable toxicity when treating lymphoid malignancies,because although effective CD19 CAR T cell therapy will result in B cellaplasia, the consequent hypogammaglobulinaemia can be treated withpooled immunoglobulin.

CD19 is therefore an attractive CAR target. To date, the main clinicalfocus of the CAR field has been studies targeting CD19 on refractoryB-cell cancers, as summarised in Table 1.

Different designs of CARs have been tested against CD19 in differentcentres, as outlined in Table 1:

TABLE 1 Summary of CAR experience targeting CD19 Centre BinderEndodomain Comment University College Fmc63 CD3-Zeta Low-level briefLondon persistence Memorial Sloane SJ25C1 CD28-Zeta Short-term Ketteringpersistence NCI/KITE Fmc63 CD28-Zeta Long-term low-level persistenceBaylor, Centre for Cell Fmc63 CD3-Zeta/ Short-term low-level and GeneTherapy CD28-Zeta persistence UPENN/Novartis Fmc63 41BB-Zeta Long-termhigh-level persistence

Most of the studies have tested CD19 CARs based on a scFv derived fromthe hybridoma fmc63. The most promising have been in the treatment ofAcute Lymphoblastic Leukaemia (ALL).

Clinical Experience with CARs Against CD19

CD19 directed CAR therapy appears most effective in ALL. The firststudies in ALL were published in Spring 2013, by groups from MemorialSloane Kettering (Brentjens, et al. (2013) Leukemia. Sci. Transl. Med.5, 177ra38) and the University of Pennsylvania. An update report of thelatter study has recently been made (Maude et al. (2014) N. Engl. J.Med. 371, 1507-1517). Here, 25 patients under the age of 25 years and 5over this age were treated. 90% achieved a complete response at onemonth, 22 of 28 evaluable cases achieved an MRD negative status and the6 month event free survival rate was 67%. 15 patients received nofurther therapy after the study.

Brentjens et al., (as above) in the adult setting, treated 5 ALLpatients (2 with refractory relapse, 2 with MRD positive disease and 1who was MRD negative) with autologous T cells retrovirally transduced toexpress a CD19 CAR incorporating an scFv derived from the SJ25C1hybridoma and a CD28 co-stimulatory domain. All of these achieved a deepmolecular remission, enabling 4 of these patients to receive anallogeneic SCT. This precluded assessment of the durability of responsesbut CAR T cells were only detectable in the blood or bone marrow for 3-8weeks after infusion. The patient who was not transplanted relapsed at90 days with CD19+ disease. Subsequently, Davila et al. ((2014). Sci.Transl. Med. 6, 224ra25) have updated this cohort. 14 of 16 adultpatients had detectable disease at the point of CAR T cell infusion,despite salvage chemotherapy and cyclophosphamide conditioning. 14 of 16achieved a complete remission with or without count recovery including 7of 9 patients with morphologic evidence of residual disease detectableafter salvage chemotherapy. 12 of 16 patients achieved MRD negativityand this allowed 7 to undergo allogeneic transplantation by the time ofpublication. Responses were durable in some patients with 4 of 8non-transplanted patients continuing in morphological remission at up to24 months follow-up although the survival curves for this cohort are notyet stable.

A recently published study in a cohort of paediatric and young adultpatients predominantly with ALL provides the first intention-to-treatanalysis of its outcomes. This may help remove the bias inherent inexcluding patients who do not receive the anticipated dose of CAR Tcells (Lee et al. (2014) Lancet. doi:10.1016/50140-6736(14)61403-3). 21patients were treated with a CD28 domain-containing second generationCAR. All but 2 patients received the anticipated T cell dose,highlighting the feasibility of delivering this treatment to those withrefractory or multiply-relapsed ALL. This study shows the followingefficacy: 67% achieving a complete remission and 60% of those with ALLachieving MRD negative status.

Immune Toxicity of CD19 CAR Therapy

Cytokine release syndrome (CRS) encompasses a range of inflammatorysymptoms ranging from mild to multi-organ failure with hypotension andrespiratory failure. Some degree of CRS occurs commonly in patientstreated with CD19 CAR T cells.

Approximately 30% (21/73) patients treated in recent cohorts showed somedegree of CRS (Davilia et al (2014) as above; Lee et al (2014) as above;Kochenderfer (2014) J. Clin. Oncol. Off. J. Am. Soc. Clin. Oncol.doi:10.1200/JCO.2014.56.2025). CRS has also been seen in patientstreated with blinatumomab, a bi-specific recombinant single-chainantibody recognising both CD19 and CD3. CRS typically occurs 5-21 daysafter CAR T cell infusion.

CRS can be life threatening and requires treatment in an intensive caresetting. CRS is associated with elevated serum cytokine levels. Thecytokines most significantly elevated are IL-6, IL-10 and interferongamma (IFNγ). Clinical manifestations of severe CRS (fever,hepatosplenomegaly, coagulopathy and hyperferritinaemia) resemblemacrophage activation syndrome (MAS) found for instance in patients withcongenital defects in T-cells. This suggests that commonimmunopathological processes are involved. At present it is not clearwhich cell type (CAR T cells, dying tumour cells, or locally-activatedmacrophages) are responsible for production of the key cytokines,particularly IL-6. However, a key initiating factor in MAS is release ofcopious Interferon-gamma (López-Alvarez et al. (2009). Clin. VaccineImmunol. CVI 16, 142-145).

Neurotoxicity

A number of patients in CD19 CAR studies across institutions havedeveloped transient neurotoxicity with a spectrum of severity fromaphasia to obtundation, delirium and seizures (Davilia et al (2014) asabove). This appears to be restricted to patients with ALL and a similarsyndrome has been documented after blinatumomab therapy. Brain imagingappears normal. Neurotoxicity may reflect high levels of systemiccytokines crossing the blood-brain barrier.

Persistence, Relapse and T-Cell Exhaustion

Durable responses appeared to correlate with higher peak levels ofcirculating CAR transduced T cells, as well as with the duration of Bcell aplasia. With exception of patients relapsing with CD19− disease,relapse was generally associated with loss of circulating CAR T cellsand recovery of normal B cells.

T cell exhaustion is a state of T cell dysfunction that arises duringmany chronic infections and cancer. It is defined by poor effectorfunction, sustained expression of inhibitory receptors and atranscriptional state distinct from that of functional effector ormemory T cells. Exhaustion prevents optimal control of infection andtumors. Recently, a clearer picture of the functional and phenotypicprofile of exhausted T cells has emerged with expression of inhibitoryreceptor programmed death 1 (PD-1; also known as PDCD1), a negativeregulator of activated T cells, being a key feature (Day et al. (2006)Nature 443, 350-354).

Responses in CD19 CAR studies suggest that persistence of T-cells for aprotracted period at high levels seems to be important in effectingdurable responses. A CD19 CAR which reduces T-cell exhaustion may resultin improved clinical responses.

There is thus a need for an alternative CAR directed against CD19 whichis not associated with the above disadvantages.

DESCRIPTION OF THE FIGURES

FIG. 1. Annotated and numbered (a) CAT19 VH sequences; (b) CAT19 VL

Sequences of the VH and VL are numbered using Chothia numbering. Theframework and CDR regions are shown. Insertions are also shown.

FIG. 2. Staining of CD19 positive cells with recombinant CAT19

SupT1 cells do not normally express CD19 but were engineered to do so inthis study. CAT19 VH and VL sequences were cloned into mouse IgG2a heavychain format and mouse kappa light chain format, both in mammalianexpression plasmids. 293T cells were transfected simultaneously withboth heavy and light chain and the resultant antibody purified withprotein A. SupT1 cells and SupT1.CD19 cells were stained with thisrecombinant antibody (or plain 293T supernatant) and further stainedwith a fluorescently conjugated anti-mouse secondary. Binding ofrecombinant CAT19 antibody could readily be detected by flow-cytometry.

FIG. 3. Staining of CD19 positive cells with CAT19 scFv

The VH and VL of CAT19 were cloned such that they form a scFv wherebythe VH and VL are separated by a (SGGGGS)₃ linker. Two scFvs weregenerated with the CAT scFv in both VH-VL and VL-VH orientations. Inaddition, scFvs were generated, also in either orientation, from theanti-CD19 antibodies fmc63 and 4g7. (a) scFv display format: this is aretroviral vector whereby the scFv is cloned onto a human IgG1 Fc spacerwhich has the CD8 transmembrane domain and the first 12 residues of theCD8 endodomain. This in turn is in frame with the FMD-2A peptide TaV andtruncated human CD34. In this way, the scFv is displayed on the surfaceof a cell, and the transgene expression can be controlled for bydetecting CD34 separately. SupT1 cells were generated which expresseither of the 6 different scFv formats and these cells were stained withrecombinant human truncated CD19-mouse IgG2a Fc fusion and anti-CD34;(b) Staining with fmc63 VH-VL and VL-VH format; (c) Staining with 4g7VH-VL and VL-VH format; and (d) Staining with CAT19 VH-VL and VL-VHformat. Surprisingly, the CAT19 VH-VL scFv bound well, while the VL-VHscFv gave significantly less detectable binding.

FIG. 4. Different generations of CARs and initial CARs tested

(a) A typical CAR format comprising of an antigen binding domain (whichmost usually is a scFv), a spacer domain, a transmembrane domain and oneor several signalling domains. (b) First generation CARs transmit anactivation signal; their endodomain is derived from either the FcGammareceptor endodomain or the CD3 Zeta endodomain; (c) Second generationreceptors transmit two signals: their endodomains comprise aco-stimulatory domain connected to the endodomain of CD3-Zeta. Theco-stimulatory domain is usually either the endodomain of CD28, theendodomain of OX40 or the endodomain of 41BB. (d) Third generationreceptors transmit three signals: their endodomains comprise a fusion ofthe CD28 endodomain with the 41BB endodomain and with the CD3-Zetaendodomain, or the CD28 endodomain with the OX40 endodomain and with theCD3-Zeta endodomain. (e) The CAT19 based CAR initially tested whichcomprises a scFv in the VH-VL orientation, a CD8 stalk spacer and 2^(nd)generation endodomain comprising of 41BB-Zeta (Campana CAR format).

FIG. 5. In vitro comparison of CAT19 CAR function against fmc36 CAR

Primary human T-cells from 5 different donors were transduced withlentiviral vectors coding for CAT19 CAR in Campana format, or theCampana CAR itself. These T-cells were then used in various assays. (a)Chromium release assay was performed against SupT1 cells. These cellsare CD19 negative. Neither CAR T-cells responded against this cell line(dotted lines). Chromium release assay was performed against SupT1.CD9.Both CARs performed equally against this cell line (unbroken lines).Next a degranulation assay was performed using either NT T-cells, fmc63CAR T-cells, or CAT19 CAR T-cells against either SupT1 or SupT1.CD19.(b) data gated on CD4+ T-cells, and (c) CD8+ T-cells is shown.Degranulation was increased with CAR19 CAR T-cells. (d) Proliferationwas estimated using tritiated thymidilation incorporation. NT, fmc63 CART-cells, CAT19 CAR T-cells were tested against SupT1. CD19. In thisexperiment, an irrelevant CAR targeting GD2 was also tested. There was atrend to increased proliferation with CAR19 CAR T-cells. (e)Interferon-gamma release from either NT T-cells, fmc63 CAR T-cells,CAT19 CAR T-cells or GD2 CAR T-cells 24 hours after challenge againstSupT1 or SupT1.CD19 cells. CAT19 CAR T-cells produced significantly lessIF-G than fmc63 CAR T-cells when challenged with CD19+ targets.

FIG. 6. In vivo model of CAT19 efficacy.

(a) Outline of experimental set-up for in vivo model. NSG mice wereinjected with 2.5×10{circumflex over ( )}5 Raji.FLuc cells via tail veininjection. 24 hours later 4×10{circumflex over ( )}6 of either NTprimary human T-cells, or T-cells transduced with fmc63 CAR, or T-cellstransduced with CAT19 CAR were administered via tail-vein. Tumourresponse was measured sequentially by bioluminescence imaging. Tail-veinblood was sampled at day 4 for engraftment and serum cytokine. Theanimals were culled at day 11 and tissues studied for persistence of CART-cells and tumour burden. (b) Bioluminescence imaging of the differentmouse cohorts at day 10. Extensive disease is seen in the pelvis, spine,ribs, skull and spleen of mice treated with NT T-cells, while minimalsignal is evident in mice who received either CAT19 CAR T-cells, orfmc63 CAR T-cells. (c) Quantitative bioluminescent signal averaged fromdifferent mouse cohorts over time. Y-axis is a log-scale; A cleardifference is seen between signal accumulation in mice who received NTT-cells, and mice who received CAR T-cells. No difference in signalaccumulation is seen in mice who received fmc63 CAR T-cells or CAT19 CART-cells. (d) Flow-cytometric determined tumour burden in bone-marrowfrom mice at the end of the experiment. Practically no Raji cells couldbe detected in marrow of mice who received either fmc63 or CAT19 CART-cells.

FIG. 7. Characterization of in vivo persisting CAR T-cells

(a) Absolute numbers of CAR T-cells in spleens of mice from animalstreated with fmc63 CAR T-cells or CAT19 CAR T-cells in the modeloutlined above. This shows the same numbers are present in both; (b)Absolute numbers of CAR T-cells in bone-marrow of mice treated withfmc63 CAR T-cells or CAT19 CAR T-cells. This shows the same numbers ofcells are present in both; (c) Absolute numbers of PD1-expressing CART-cells in spleen and (d) bone-marrow of mice treated with either fmc63CAR T-cells or CAT19 CAR T-cells. Fewer of the CAT19 T-cells are PD1+ inboth compartments.

SUMMARY OF ASPECTS OF THE INVENTION

The present inventors have developed a new CD19-specific CAR with CDRsthat have not previously been described. It has equivalent potency tothe fmc63-based CAR used in the UPENN studies, but results in reducedtoxicity and reduced T-cell exhaustion.

Thus, in a first aspect the present invention provides a chimericantigen receptor (CAR) comprising a CD19-binding domain which comprisesa) a heavy chain variable region (VH) having complementarity determiningregions (CDRs) with the following sequences:

CDR1 (SEQ ID No. 1) GYAFSSS; CDR2 (SEQ ID No. 2) YPGDED CDR3 (SEQ ID No.3) SLLYGDYLDY;andb) a light chain variable region (VL) having CDRs with the followingsequences:

CDR1 (SEQ ID No. 4) SASSSVSYMH; CDR2 (SEQ ID No. 5) DTSKLAS CDR3 (SEQ IDNo. 6) QQWNINPLT.

The CD19 binding domain may comprise a VH domain having the sequenceshown as SEQ ID No. 7 and/or or a VL domain having the sequence shown asSEQ ID No 8 or a variant thereof having at least 95% sequence identity.

The CD19 binding domain may comprise an scFv in the orientation VH-VL.

The CD19 binding domain may comprise the sequence shown as SEQ ID No 9or a variant thereof having at least 90% sequence identity.

The CD19 binding domain may comprise the 6 CDRs defined in claim 1grafted on to a human antibody framework.

The CD19-binding domain and the transmembrane domain may be connected bya spacer, which may comprise one of the following: a human an IgG1 Fcdomain; an IgG1 hinge; or a CD8 stalk. The spacer may comprise a CD8stalk.

The CAR may comprise or associate with an intracellular T cellsignalling domain.

The intracellular T cell signalling domain may comprise one or more ofthe following endodomains: CD28 endodomain; 41BB endodomain, OX40endodomain and the CD3-Zeta endodomain.

In particular the CAR may comprise a CD8 stalk spacer and anintracellular T-cell signalling domain which comprises the 41BBendodomain and the CD3-Zeta endodomain.

In particular the CAR may comprise a CD8 stalk spacer and anintracellular T-cell signalling domain which comprises the OX40endodomain and the CD3-Zeta endodomain.

In an alternative embodiment, the intracellular T cell signalling domainmay comprise all of the following endodomains: CD28 endodomain; OX40 andCD3-Zeta endodomain.

The CAR may comprise the sequence shown as any of SEQ ID No. 10 to 15 ora variant thereof which has at least 80% sequence identity but retainsthe capacity to i) bind CD19 and ii) induce T cell signalling. The CARmay have advantageous properties compared to the fmc63-based CAR used inthe UPENN studies. For example, the CAR, when expressed by a T-cell andused to target a CD19 expressing cell, may cause lower IFNγ release bythe CD19-expressing target cell than that caused by a T-cell expressinga CAR comprising a CD19-binding domain which comprises: a) a heavy chainvariable region (VH) having complementarity determining regions (CDRs)with the following sequences:CDR1—GVSLPDY (SEQ ID No. 16); CDR2—WGSET(SEQ ID No. 17); CDR3—HYYYGGSYAMDY (SEQ ID No. 18); and b) a light chainvariable region (VL) having CDRs with the followingsequences:CDR1—RASQDISKYLN (SEQ ID No. 19); CDR2—HTSRLHS (SEQ ID No. 20)CDR3—QQGNTLPYT (SEQ ID No. 21). The CDRs may be grafted on to a human orhumanised framework.

In a second aspect, the present invention provides a nucleic acidsequence which encodes a CAR according to the first aspect of theinvention.

In a third aspect, there is provided a vector which comprises a nucleicacid sequence according to the second aspect of the invention.

In a third aspect there is provided a cell which comprises a CARaccording to the first aspect of the invention.

The cell may be a cytolytic immune cell, such as a T cell or a naturalkiller (NK) cell.

In a fourth aspect there is provided a cell composition which comprisesa plurality of cells according to the third aspect of the invention.

In a fifth aspect, there is provided a method for making a cellaccording to the third aspect of the invention, which comprises the stepof transducing or transfecting a cell with a vector according to thethird aspect of the invention.

In a sixth aspect there is provided a method for making a cellcomposition according to the fourth aspect of the invention whichcomprises the step of transducing or transfecting a sample of cells froma subject ex vivo with a vector according to the third aspect of theinvention.

The sample of cells may, for example, be a blood sample or a derivativethereof, such as a peripheral blood mononuclear cell (PBMC) sample.

In a seventh aspect, there is provided a pharmaceutical compositionwhich comprises a cell according to the first aspect of the invention,or a cell composition according to the fourth aspect of the invention,together with a pharmaceutically acceptable carrier, diluent orexcipient.

In an eighth aspect, there is provided a method for treating cancerwhich comprises the step of administering a cell according to the firstaspect of the invention, a cell composition according to the fourthaspect of the invention or a pharmaceutical composition according to theseventh aspect of the invention to a subject.

The method may comprise the step of transducing or transfecting cellsfrom the subject ex vivo with a vector according to the third aspect ofthe invention, then administering the, or some of the, transfected cellsback to the subject.

There is also provided a pharmaceutical composition according to theseventh aspect of the invention for use in treating cancer.

There is also provided the use of a cell according to the third aspectof the invention in the manufacture of a pharmaceutical composition fortreating cancer.

The cancer may, for example, be a B cell malignancy.

DETAILED DESCRIPTION

Chimeric Antigen Receptors (CARs)

Chimeric antigen receptors (CARs), also known as chimeric T cellreceptors, artificial T cell receptors and chimeric immunoreceptors, areengineered receptors, which graft an arbitrary specificity onto animmune effector cell. In a classical CAR, the specificity of amonoclonal antibody is grafted on to a T cell. CAR-encoding nucleicacids may be transferred to T cells using, for example, retroviralvectors. In this way, a large number of cancer-specific T cells can begenerated for adoptive cell transfer. Phase I clinical studies of thisapproach show efficacy.

The target-antigen binding domain of a CAR is commonly fused via aspacer and transmembrane domain to an endodomain. The endodomain maycomprise or associate with an intracellular T-cell signalling domain.When the CAR binds the target-antigen, this results in the transmissionof an activating signal to the T-cell it is expressed on.

The CAR of the present invention comprises a CD19 binding domain whichis based on a mouse anti-CD19 monoclonal antibody.

The CAR of the present invention comprises a CD19-binding domain whichcomprises

-   -   a) a heavy chain variable region (VH) having complementarity        determining regions (CDRs) with the following sequences:

CDR1 (SEQ ID No. 1) GYAFSSS; CDR2 (SEQ ID No. 2) YPGDED CDR3 (SEQ ID No.3) SLLYGDYLDY;and

-   -   b) a light chain variable region (VL) having CDRs with the        following sequences:

CDR1 (SEQ ID No. 4) SASSSVSYMH; CDR2 (SEQ ID No. 5) DTSKLAS CDR3(SEQ ID No. 6) QQWNINPLT.

It may be possible to introduce one or more mutations (substitutions,additions or deletions) into each CDR without negatively affectingCD19-binding activity. Each CDR may, for example, have one, two or threeamino acid mutations.

The CDRs may be in the format of a single-chain variable fragment(scFv), which is a fusion protein of the heavy variable region (VH) andlight chain variable region (VL) of an antibody, connected with a shortlinker peptide of ten to about 25 amino acids. The scFv may be in theorientation VH-VL, i.e. the VH is at the amino-terminus of the CARmolecule and the VL domain is linked to the spacer and, in turn thetransmembrane domain and endodomain.

The CDRs may be grafted on to the framework of a human antibody or scFv.For example, the CAR of the present invention may comprise aCD19-binding domain consisting or comprising one of the followingsequences

The CAR of the present invention may comprise the following VH sequence:

VH sequence from murine monoclonal antibody SEQ ID No. 7QVQLQQSGPELVKPGASVKISCKASGYAFSSSWMNWVKQRPGKGLEWIGRIYPGDEDTNYSGKFKDKATLTADKSSTTAYMQLSSLTSEDSAVYFCARSL LYGDYLDYWGQGTTLTVSS

The CAR of the present invention may comprise the following VL sequence:

VL sequence from murine monoclonal antibody SEQ ID No 8QIVLTQSPAIMSASPGEKVTMTCSASSSVSYMHWYQQKSGTSPKRWIYDTSKLASGVPDRFSGSGSGTSYFLTINNMEAEDAATYYCQQWNINPLTFGAG TKLELKR

The CAR of the invention may comprise the following scFv sequence:

VH-VL scFv sequence from murine monoclonal antibody SEQ ID No 9QVQLQQSGPELVKPGASVKISCKASGYAFSSSWMNWVKQRPGKGLEWIGRIYPGDEDTNYSGKFKDKATLTADKSSTTAYMQLSSLTSEDSAVYFCARSLLYGDYLDYWGQGTTLTVSSGGGGSGGGGSGGGGSQIVLTQSPAIMSASPGEKVTMTCSASSSVSYMHWYQQKSGTSPKRWIYDTSKLASGVPDRFSGSGSGTSYFLTINNMEAEDAATYYCQQWNINPLTFGAGTKLELKR

The CAR may consist of or comprise one of the following sequences:

CAT19 CAR using “Campana” architecture (see Examples) SEQ ID No. 10MGTSLLCWMALCLLGADHADAQVQLQQSGPELVKPGASVKISCKASGYAFSSSWMNWVKQRPGKGLEWIGRIYPGDEDTNYSGKFKDKATLTADKSSTTAYMQLSSLTSEDSAVYFCARSLLYGDYLDYWGQGTTLTVSSGGGGSGGGGSGGGGSQIVLTQSPAIMSASPGEKVTMTCSASSSVSYMHWYQQKSGTSPKRWIYDTSKLASGVPDRFSGSGSGTSYFLTINNMEAEDAATYYCQQWNINPLTFGAGTKLELKRSDPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRCAT19 CAR with an OX40-Zeta endodomain SEQ ID No. 11MGTSLLCWMALCLLGADHADAQVQLQQSGPELVKPGASVKISCKASGYAFSSSWMNWVKQRPGKGLEWIGRIYPGDEDTNYSGKFKDKATLTADKSSTTAYMQLSSLTSEDSAVYFCARSLLYGDYLDYWGQGTTLTVSSGGGGSGGGGSGGGGSQIVLTQSPAIMSASPGEKVTMTCSASSSVSYMHWYQQKSGTSPKRWIYDTSKLASGVPDRFSGSGSGTSYFLTINNMEAEDAATYYCQQWNINPLTFGAGTKLELKRSDPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCRRDQRLPPDAHKPPGGGSFRTPIQEEQADAHSTLAKIRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR CAT19 CAR with a CD28-Zeta endodomainSEQ ID No. 12 MGTSLLCWMALCLLGADHADAQVQLQQSGPELVKPGASVKISCKASGYAFSSSWMNWVKQRPGKGLEWIGRIYPGDEDTNYSGKFKDKATLTADKSSTTAYMQLSSLTSEDSAVYFCARSLLYGDYLDYWGQGTTLTVSSGGGGSGGGGSGGGGSQIVLTQSPAIMSASPGEKVTMTCSASSSVSYMHWYQQKSGTSPKRWIYDTSKLASGVPDRFSGSGSGTSYFLTINNMEAEDAATYYCQQWNINPLTFGAGTKLELKRSDPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR Third generation CD19 CARSEQ ID No. 13 MGTSLLCWMALCLLGADHADAQVQLQQSGPELVKPGASVKISCKASGYAFSSSWMNWVKQRPGKGLEWIGRIYPGDEDTNYSGKFKDKATLTADKSSTTAYMQLSSLTSEDSAVYFCARSLLYGDYLDYWGQGTTLTVSSGGGGSGGGGSGGGGSQIVLTQSPAIMSASPGEKVTMTCSASSSVSYMHWYQQKSGTSPKRWIYDTSKLASGVPDRFSGSGSGTSYFLTINNMEAEDAATYYCQQWNINPLTFGAGTKLELKRSDPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRDQRLPPDAHKPPGGGSFRTPIQEEQADAHSTLAKIRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR CD19 CAR with IgG1 hinge spacerSEQ ID No. 14 MGTSLLCWMALCLLGADHADAQVQLQQSGPELVKPGASVKISCKASGYAFSSSWMNWVKQRPGKGLEWIGRIYPGDEDTNYSGKFKDKATLTADKSSTTAYMQLSSLTSEDSAVYFCARSLLYGDYLDYWGQGTTLTVSSGGGGSGGGGSGGGGSQIVLTQSPAIMSASPGEKVTMTCSASSSVSYMHWYQQKSGTSPKRWIYDTSKLASGVPDRFSGSGSGTSYFLTINNMEAEDAATYYCQQWNINPLTFGAGTKLELKRSDPAEPKSPDKTHTCPPCPKDPKFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTAT KDTYDALHMQALPPRCD19 CAR with hinge-CH2—CH3 of humanIgG1 with FcR binding sites mutated out SEQ ID No. 15MGTSLLCWMALCLLGADHADAQVQLQQSGPELVKPGASVKISCKASGYAFSSSWMNWVKQRPGKGLEWIGRIYPGDEDTNYSGKFKDKATLTADKSSTTAYMQLSSLTSEDSAVYFCARSLLYGDYLDYWGQGTTLTVSSGGGGSGGGGSGGGGSQIVLTQSPAIMSASPGEKVTMTCSASSSVSYMHWYQQKSGTSPKRWIYDTSKLASGVPDRFSGSGSGTSYFLTINNMEAEDAATYYCQQWNINPLTFGAGTKLELKRSDPAEPKSPDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMIARTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKKDPKFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

The CAR of the invention may comprise a variant of the sequence shown asSEQ ID No. 7, 8, 9, 10, 11, 12, 13, 14 or 15 having at least 80, 85, 90,95, 98 or 99% sequence identity, provided that the variant sequenceretain the capacity to bind CD19 (when in conjunction with acomplementary VL or VH domain, if appropriate).

The percentage identity between two polypeptide sequences may be readilydetermined by programs such as BLAST which is freely available athttp://blast.ncbi.nlm.nih.gov.

Transmembrane Domain

The CAR of the invention may also comprise a transmembrane domain whichspans the membrane. It may comprise a hydrophobic alpha helix. Thetransmembrane domain may be derived from CD28, which gives good receptorstability.

The transmembrane domain may comprise the sequence shown as SEQ ID No.22.

SEQ ID No. 22 FWVLVVVGGVLACYSLLVTVAFIIFWVIntracellular T Cell Signaling Domain (Endodomain)

The endodomain is the signal-transmission portion of the CAR. Afterantigen recognition, receptors cluster and a signal is transmitted tothe cell. The most commonly used endodomain component is that ofCD3-zeta which contains 3 ITAMs. This transmits an activation signal tothe T cell after antigen is bound. CD3-zeta may not provide a fullycompetent activation signal and additional co-stimulatory signaling maybe needed. For example, endodomains from CD28, or OX40 or 41BB can beused with CD3-Zeta to transmit a proliferative/survival signal, or allthree can be used together.

Early CAR designs had endodomains derived from the intracellular partsof either the γ chain of the FcεR1 or CD3ζ. Consequently, these firstgeneration receptors transmitted immunological signal 1, which wassufficient to trigger T-cell killing of cognate target cells but failedto fully activate the T-cell to proliferate and survive. To overcomethis limitation, compound endodomains were constructed. Fusion of theintracellular part of a T-cell co-stimulatory molecule to that of CD3ζresulted in second generation receptors which could transmit anactivating and co-stimulatory signal simultaneously after antigenrecognition. The co-stimulatory domain most commonly used was that ofCD28. This supplies the most potent co-stimulatory signal, namelyimmunological signal 2, which triggers T-cell proliferation. Somereceptors were also described which included TNF receptor familyendodomains such as OX40 and 41BB which transmit survival signals.Finally, even more potent third generation CARs were described which hadendodomains capable of transmitting activation, proliferation andsurvival signals. CARs and their different generations are summarized inFIG. 4.

The endodomain of the CAR of the present invention may comprisecombinations of one or more of the CD3-Zeta endodomain, the 41BBendodomain, the OX40 endodomain or the CD28 endodomain.

The intracellular T-cell signalling domain (endodomain) of the CAR ofthe present invention may comprise the sequence shown as SEQ ID No. 23,24, 25, 26, 27, 28, 29 or 30 or a variant thereof having at least 80%sequence identity.

(CD3 zeta endodomain) SEQ ID No. 23RSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATK DTYDALHMQALPPR (41BBendodomain) SEQ ID No. 24 KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL(OX40 endodomain) SEQ ID No. 25 RRDQRLPPDAHKPPGGGSFRTPIQEEQADAHSTLAKI(CD28 endodomain) SEQ ID No. 26 KRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAY

Examples of combinations of such endodomains include 41BB-Z, OX40-Z,CD28-Z and CD28-OX40-Zeta.

(41BB-Z endodomain fusion) SEQ ID No. 27KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQA LPPR(OX40-Z endodomain fusion) SEQ ID No. 28RRDQRLPPDAHKPPGGGSFRTPIQEEQADAHSTLAKIRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(CD28Z endodomain fusion) SEQ ID No. 29KRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPP R (CD28OXZ)SEQ ID No. 30 KRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRDQRLPPDAHKPPGGGSFRTPIQEEQADAHSTLAKIRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

A variant sequence may have at least 80%, 85%, 90%, 95%, 98% or 99%sequence identity to SEQ ID No. 22, 23, 24, 25, 26, 27, 28, 29 or 30provided that the sequence provides an effective transmembranedomain/intracellular T cell signaling domain.

Signal Peptide

The CAR of the present invention may comprise a signal peptide so thatwhen the CAR is expressed inside a cell, such as a T-cell, the nascentprotein is directed to the endoplasmic reticulum and subsequently to thecell surface, where it is expressed.

The core of the signal peptide may contain a long stretch of hydrophobicamino acids that has a tendency to form a single alpha-helix. The signalpeptide may begin with a short positively charged stretch of aminoacids, which helps to enforce proper topology of the polypeptide duringtranslocation. At the end of the signal peptide there is typically astretch of amino acids that is recognized and cleaved by signalpeptidase. Signal peptidase may cleave either during or after completionof translocation to generate a free signal peptide and a mature protein.The free signal peptides are then digested by specific proteases.

The signal peptide may be at the amino terminus of the molecule.

The CAR of the invention may have the general formula:Signal peptide-CD19-binding domain-spacer domain-transmembranedomain/intracellular T cell signaling domain.

The signal peptide may comprise the SEQ ID No. 31 or a variant thereofhaving 5, 4, 3, 2 or 1 amino acid mutations (insertions, substitutionsor additions) provided that the signal peptide still functions to causecell surface expression of the CAR.

SEQ ID No. 31: METDTLLLWVLLLWVPGSTG

The signal peptide of SEQ ID No. 31 is compact and highly efficient. Itis predicted to give about 95% cleavage after the terminal glycine,giving efficient removal by signal peptidase.

Spacer

The CAR of the present invention may comprise a spacer sequence toconnect the CD19-binding domain with the transmembrane domain andspatially separate the CD19-binding domain from the endodomain. Aflexible spacer allows to the CD19-binding domain to orient in differentdirections to enable CD19 binding.

The spacer sequence may, for example, comprise an IgG1 Fc region, anIgG1 hinge or a CD8 stalk, or a combination thereof. The spacer mayalternatively comprise an alternative sequence which has similar lengthand/or domain spacing properties as an IgG1 Fc region, an IgG1 hinge ora CD8 stalk.

A human IgG1 spacer may be altered to remove Fc binding motifs.

Examples of amino acid sequences for these spacers are given below:

(hinge-CH2CH3 of human IgG1) SEQ ID No. 32AEPKSPDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMIARTPEVTCVVVDVSHEDPEVKFNVVYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKKD (human CD8 stalk): SEQ ID No. 33TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDI (human IgG1 hinge):SEQ ID No. 34 AEPKSPDKTHTCPPCPKDPK (IgG1 Hinge-Fc) SEQ ID No. 35AEPKSPDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKKDPK(IgG1 Hinge - Fc modified to remove Fc receptor recognition motifs)SEQ ID No. 36 AEPKSPDKTHTCPPCPAPPVA*GPSVFLFPPKPKDTLMIARTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKKDPK

Modified residues are underlined; * denotes a deletion.

Interferon Release and Car T-Cell Exhaustion

The present inventors have found that a CD19 CAR based on the CAT19 scFvhas properties which may result in lower toxicity and better efficacy.

Given that the main experience with CD19 CAR therapy has been with CARsbased on the fmc63 scFv, and that the oldest, largest and perhaps mostsignificant clinical data set is with the fmc63 based Campana CAR, thepresent inventors took this Campana CAR as the “gold-standard”. Acomparison was hence made between the fmc63-Campana CAR and a similarCAR but with CAT19 scFv instead of fmc63. Surprisingly, the presentinventors found that while CAT19 CAR T-cells effected killing of targetcell expressing CD19, and proliferated in response to CD19 expressingtargets, Interferon-gamma release was less. Further, a small animalmodel of an aggressive B-cell lymphoma showed equal efficacy and equalengraftment between the fmc63 and CAT19 based CARs, but surprisingly,less of the CAT19 CAR T-cells were exhausted than fmc63 CAR T-cells.

The CAR of the invention may cause 25, 50, 70 or 90% lower IFNγ releasein a comparative assay involving bringing CAR T cells into contact withtarget cells.

The CAR of the invention may result in a smaller proportion of CAR Tcells becoming exhausted than fmc63 CAR T cells. T cell exhaustion maybe assessed using methods known in the art, such as analysis of PD-1expression. The CAR of the present invention may cause 20, 30, 40, 50,60 of 70% fewer CAR T cells to express PD-1 that fmc63 CAR T cells in acomparative assay involving bringing CAR T cells into contact withtarget cells.

Nucleic Acid Sequence

The second aspect of the invention relates to a nucleic acid sequencewhich codes for a CAR of the first aspect of the invention.

The nucleic acid sequence may be capable of encoding a CAR having theamino acid sequence shown as any of SEQ ID No. 10-15.

Vector

The present invention also provides a vector which comprises a nucleicacid sequence according to the present invention. Such a vector may beused to introduce the nucleic acid sequence into a host cell so that itexpresses and produces a molecule according to the first aspect of theinvention.

The vector may, for example, be a plasmid or a viral vector, such as aretroviral vector or a lentiviral vector.

The vector may be capable of transfecting or transducing a cell, such asa T cell.

Cell

The invention also provides a cell which comprises a nucleic acidaccording to the invention. The invention provides a cell whichexpresses a CAR according to the first aspect of the invention at thecell surface.

The cell may be a cytolytic immune cell, such as a T-cell or naturalkiller (NK) cell.

A cell capable of expressing a CAR according to the invention may bemade by transducing or transfecting a cell with CAR-encoding nucleicacid.

The CAR-expressing cell of the invention may be generated ex vivo. Thecell may be from a cell sample, such as a peripheral blood mononuclearcell (PBMC) sample from the patient or a donor. Cells may be activatedand/or expanded prior to being transduced with CAR-encoding nucleicacid, for example by treatment with an anti-CD3 monoclonal antibody.

Pharmaceutical Composition

The present invention also relates to a pharmaceutical compositioncontaining a CAR-expressing cell, or plurality of cells, of theinvention together with a pharmaceutically acceptable carrier, diluentor excipient, and optionally one or more further pharmaceutically activepolypeptides and/or compounds. Such a formulation may, for example, bein a form suitable for intravenous infusion.

Method of Treatment

CAR-expressing cells of the present invention may be capable of killingcancer cells, such as B-cell lymphoma cells. CAR-expressing cells, suchas T-cells or NK cells, may either be created ex vivo either from apatient's own peripheral blood (1^(st) party), or in the setting of ahaematopoietic stem cell transplant from donor peripheral blood (2^(nd)party), or peripheral blood from an unconnected donor (3^(rd) party).Alternatively, CAR-expressing cells may be derived from ex vivodifferentiation of inducible progenitor cells or embryonic progenitorcells to cells such as T-cells. In these instances, CAR cells aregenerated by introducing DNA or RNA coding for the CAR by one of manymeans including transduction with a viral vector, transfection with DNAor RNA.

T or NK cells expressing a CAR molecule of the present invention may beused for the treatment of a cancerous disease, in particular a cancerousdisease associated with CD19 expression.

A method for the treatment of disease relates to the therapeutic use ofa cell or population of cells of the invention. In this respect, thecells may be administered to a subject having an existing disease orcondition in order to lessen, reduce or improve at least one symptomassociated with the disease and/or to slow down, reduce or block theprogression of the disease. The method of the invention may cause orpromote cell mediated killing of CD19-expressing cells, such as B cells.

The invention will now be further described by way of Examples, whichare meant to serve to assist one of ordinary skill in the art incarrying out the invention and are not intended in any way to limit thescope of the invention.

EXAMPLES Example 1—Cloning of VH and VL and Demonstration of CD19Binding

The VH and VL were cloned from a mouse anti-CD19 monoclonal antibody andfused in frame with the human kappa constant region and the human IgG1constant region. These chimeric heavy and light chains were cloned intoan expression vector and used to transfect 293T cells. The subsequentproduced antibody was used to stain SupT1 cells (a T-cell line which isCD19 negative), and SupT1 cells which have been engineered to be CD19positive. This staining shows specific binding of the CD19 (FIG. 2).

Example 2—Demonstration that the VH/VL can Form an scFv which Binds CD19

It was then investigated whether the cloned VH and VL could bind CD19 ina scFv format. The VH and VL were cloned as an scFv in two orientations:VH-VL and VL-VH, where the two variable regions were separated by alinker comprising of (SGGGG)4. These scFv were cloned into anon-signalling CAR co-expressed with truncated CD34 as shown in FIG. 3a. Briefly, this comprises of a signal peptide, scFv, hinge-CH2-CH3 ofhuman IgG1, the CD8 transmembrane domain, the first 12 residues of theCD8 endodomain, a FMD-2A peptide TeV, truncated human CD34. To allowcomparison, scFv from fmc63, and scFv from another anti-CD19 hybridoma4g7, were cloned in the same format in both VH-VL and VL-VHorientations.

In this way, several parameters can be studied: (1) the binding oftarget antigen to the CAR by use of recombinant cognate target antigenfused to murine Fc, unencumbered by internalization of the receptor dueto signalling; (2) The stability of the receptor can be determined usingpolyclonal anti-Fc; (3) the expression levels of the cassette can becontrolled for by co-staining for CD34.

These constructs were transduced into SupT1 cells. RecombinantCD19-mouse IgG2aFc fusion was generated. SupT1 cells were stained formouse-Fc, human-Fc and anti-CD34 with antibodies conjugated to differentfluorophores and stability/binding interrogated by flow-cytometery.

The sequences of the different scFvs used are detailed below:

>scFv_fmc63_VH-VL (SEQ ID No. 37)EVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSGGGGSGGGGSGGGGSDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITKA >scFv_fmc63_VL-VH(SEQ ID No. 38) DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITKAGGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSS >scFv_4g7_VH-VL(SEQ ID No. 39) EVQLQQSGPELIKPGASVKMSCKASGYTFTSYVMHWVKQKPGQGLEWIGYINPYNDGTKYNEKFKGKATLTSDKSSSTAYMELSSLTSEDSAVYYCARGTYYYGSRVFDYWGQGTTLTVSSGGGGSGGGGSGGGGSDIVMTQAAPSIPVTPGESVSISCRSSKSLLNSNGNTYLYWFLQRPGQSPQLLIYRMSNLASGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYCMQHLEYPFTFGAGTKLELKR >scFv_4g7_VL-VH(SEQ ID No. 40) DIVMTQAAPSIPVTPGESVSISCRSSKSLLNSNGNTYLYWFLQRPGQSPQLLIYRMSNLASGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYCMQHLEYPFTFGAGTKLELKRSGGGGSGGGGSGGGGSEVQLQQSGPELIKPGASVKMSCKASGYTFTSYVMHWVKQKPGQGLEWIGYINPYNDGTKYNEKFKGKATLTSDKSSSTAYMELSSLTSEDSAVYYCARGTYYYGSRVFDYWGQGTTLTVSS >scFv_CAT_VH-VL(SEQ ID No. 9) QVQLQQSGPELVKPGASVKISCKASGYAFSSSWMNWVKQRPGKGLEWIGRIYPGDEDTNYSGKFKDKATLTADKSSTTAYMQLSSLTSEDSAVYFCARSLLYGDYLDYWGQGTTLTVSSGGGGSGGGGSGGGGSQIVLTQSPAIMSASPGEKVTMTCSASSSVSYMHWYQQKSGTSPKRWIYDTSKLASGVPDRFSGSGSGTSYFLTINNMEAEDAATYYCQQWNINPLTFGAGTKLELKR >scFv_CAT_VL-VH(SEQ ID No. 41) QIVLTQSPAIMSASPGEKVTMTCSASSSVSYMHWYQQKSGTSPKRWIYDTSKLASGVPDRFSGSGSGTSYFLTINNMEAEDAATYYCQQWNINPLTFGAGTKLELKRSGGGGSGGGGSGGGGSQVQLQQSGPELVKPGASVKISCKASGYAFSSSWMNWVKQRPGKGLEWIGRIYPGDEDTNYSGKFKDKATLTADKSSTTAYMQLSSLTSEDSAVYFCARSLLYGDYLDYWGQGTTLTVSS

The construct used and the staining results are summarized in FIG. 3.Surprisingly, the CAT CAR with scFv in VH-VL orientation binds CD19,while the CAT19 CAR with scFv in the VL-VH orientation gave minimal CD19binding. This was in contrast to the fmc63 CARs and 4g7 CARs which boundCD19 in both the HL and LH orientations. Binding and stability of the HLCAT CAR appeared equal to that with fmc63.

Example 3—In Vitro Comparison of CAT19 CAR Function Against Fmc36 CAR

The CAT scFv in HL orientation was cloned into a CAR scaffold designedby Campana (Imai et al (2004) Leuk. Off. J. Leuk. Soc, Am. Leuk, Res.Fund. UK 18:676-684). Effectively the fmc63 scFv was replaced with a CATscFv, and compared with the original fmc63 based CAR. This CAR comprisesa signal peptide, the scFv, a CD8 stalk spacer and transmembrane and41BB and Zeta endodomains. The amino acid sequences of the CAT CAR andfmc63 CAR are given below:

>CAT19_CAR (SEQ ID No. 10)MGTSLLCWMALCLLGADHADAQVQLQQSGPELVKPGASVKISCKASGYAFSSSWMNWVKQRPGKGLEWIGRIYPGDEDTNYSGKFKDKATLTADKSSTTAYMQLSSLTSEDSAVYFCARSLLYGDYLDYWGQGTTLTVSSGGGGSGGGGSGGGGSQIVLTQSPAIMSASPGEKVTMTCSASSSVSYMHWYQQKSGTSPKRWIYDTSKLASGVPDRFSGSGSGTSYFLTINNMEAEDAATYYCQQWNINPLTFGAGTKLELKRSDPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR >Fmc63_CAR, as described byImai et al (2004) as above (SEQ ID No. 42)METDTLLLWVLLLWVPGSTGDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITKAGGGGSGGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSDPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

Primary human T-cells from 5 different donors were transduced withlentiviral vectors coding for CAT19 CAR in Campana format, or the fmc63Campana CAR itself. These T-cells were then used in various assays.Chromium release assay was performed against SupT1 cells. These cellsare CD19 negative. Neither CAR T-cells responded against this cell linedemonstrating that CAR19 CAR has no non-specific killing activityagainst CD19 negative cells [FIG. 5(a)]. (b) Chromium release assay wasalso performed against SupT1 cells engineering to express CD19. BothCARs performed equally against this cell line in this assay withhigh-levels of killing [FIG. 5(b)]. Next a degranulation assay wasperformed by staining for CD107 on the surface of effector cells afterco-culture with target cells. Here either NT T-cells, fmc63 CAR T-cells,or CAT19 CAR T-cells were used as effectors and either SupT1 orSupT1.CD19 cells were used as targets. Surface CD107 was detected byflow-cytometry which allowed differential measurement of degranulationof CD4+ and CD8+ cells. [FIGS. 5(c) and (d) respectively]. Degranulationwas increased with CAT19 CAR T-cells in comparison with fmc63 CART-cells. Proliferation was estimated using tritiated thymidilateincorporation. Here, NT, fmc63 CAR T-cells, CAT19 CAR T-cells wereco-cultured with SupT1 cells engineered to express CD19. Incorporationof thymidiln this experiment, an irrelevant CAR targeting GD2 was alsotested. There was a trend to increased proliferation with CAR19 CART-cells [FIG. 4(e)]. Next, interferon-gamma release from either NTT-cells, fmc63 CAR T-cells, CAT19 CAR T-cells or GD2 CAR T-cells 24hours after challenge against SupT1 or SupT1.CD19 cells was measured byELISA. CAT19 CAR T-cells produced significantly less interferon-gammathan fmc63 CAR T-cells when challenged with CD19+ targets.

Example 4—Demonstration of In Vivo Efficacy of CAT19 CAR Therapy

An outline of experimental set-up for this in vivo model is present inFIG. 6(a). Briefly NSG (NOD scid gamma, NOD.Cg-Prkdc^(scid)II2rg^(tm1Wjl)/SzJ) mice are sufficiently immunocompromised that theyare permissive for engraftment of human cell lines and primary humanT-cells. Raji cells are a B-cell line derived from Burkitt's lymphoma.These cells readily engraft within the bone-marrow of NSG mice causingan aggressive leukaemia-like syndrome. Raji cells were engineered toexpress fire-fly Luciferase to allow non-invasive tracking usingbioluminescence imaging (BLI). Mice were injected with 2.5×10{circumflexover ( )}5 Raji.FLuc cells via tail vein injection. 24 hours later4×10{circumflex over ( )}6 of either NT primary human T-cells, orT-cells transduced with fmc63 CAR, or T-cells transduced with CAT19 CARwere administered via tail-vein. Tumour response was measuredsequentially by BLI. Tail-vein blood was sampled at day 4 forengraftment and serum cytokine. The animals were culled at day 11 andtissues studied for persistence of CAR T-cells and tumour burden. BLIimaging of the different mouse cohorts at day 10 is shown in FIG. 6(b).Extensive disease is seen in the pelvis, spine, ribs, skull and spleenof mice treated with NT T-cells, while minimal signal is evident in micewho received either CAT19 CAR T-cells, or fmc63 CAR T-cells.Quantitative bioluminescent signal averaged from different mouse cohortsover time is shown on a log-scale in FIG. 6(c). A clear difference isseen between signal accumulation in mice who received NT T-cells, andmice who received CAR T-cells. No difference in signal accumulation isseen in mice who received fmc63 CAR T-cells or CAT19 CAR T-cells.Finally, after sacrifice, flow-cytometric analysis of bone-marrow fromeach mouse was performed to directly determine tumour burden. Raji cellsare easily distinguishable from mouse haematopoietic cells and fromadoptively transferred T-cells, since they express human B-cell markers.Minimal Raji cells could be detected in marrow of mice who receivedeither fmc63 or CAT19 CAR T-cells.

Example 5—Characterization of In Vivo Persisting CAR T-Cells

From the above animal models, the present inventors sought to determineif both types of CAR T-cells engrafted within the bone-marrow and spleenof these NSG mice. Flow-cytometric analysis of bone-marrow and spleenwith counting beads allowed determination of absolute numbers of CART-cells. This data is shown in FIGS. 7(a) and (b). The absolute numbersof CAR T-cells in spleens of mice from animals treated with fmc63 CART-cells or CAT19 CAR T-cells was similar. Next, the present inventorsproceeded to determine if there was any difference in the numbers ofexhausted T-cells in these different tissues. By co-staining for PD1expression in the above samples the numbers of exhausted T-cells couldbe determined. This data is shown in FIGS. 7(c) and (d). Surprisingly,fewer exhausted T-cells were present in both tissue compartments withthe CAT19 CAR than the fmc63 CAR.

All publications mentioned in the above specification are hereinincorporated by reference. Various modifications and variations of thedescribed methods and system of the invention will be apparent to thoseskilled in the art without departing from the scope and spirit of theinvention. Although the invention has been described in connection withspecific preferred embodiments, it should be understood that theinvention as claimed should not be unduly limited to such specificembodiments. Indeed, various modifications of the described modes forcarrying out the invention which are obvious to those skilled inmolecular biology, CAR technology or related fields are intended to bewithin the scope of the following claims.

The invention claimed is:
 1. A chimeric antigen receptor (CAR)comprising a CD19-binding domain which comprises: a) a heavy chainvariable region (VH) having complementarity determining regions (CDRs)with the following sequences: CDR1 (SEQ ID NO. 1) GYAFSSS; CDR2(SEQ ID NO. 2) YPGDED; and CDR3 (SEQ ID NO. 3) SLLYGDYLDY;

and b) a light chain variable region (VL) having CDRs with the followingsequences: CDR1 (SEQ ID NO. 4) SASSSVSYMH; CDR2 (SEQ ID NO. 5) DTSKLAS;and CDR3 (SEQ ID NO. 6) QQWNINPLT.


2. The CAR according to claim 1, wherein the CD19 binding domaincomprises a VH domain having the sequence shown as SEQ ID NO: 7 and/oror a VL domain having the sequence shown as SEQ ID NO: 8 or a variantthereof having at least 95% sequence identity.
 3. The CAR according toclaim 1, wherein the CD19 binding domain comprises an scFv in theorientation VH-VL.
 4. The CAR according to claim 3, wherein the CD19binding domain comprises the sequence shown as SEQ ID NO: 9 or a variantthereof having at least 90% sequence identity.
 5. The CAR according toclaim 1, wherein CD19-binding domain is connect to a transmembranedomain by a spacer.
 6. The CAR according to claim 5, wherein the spacercomprises one of the following: a human an IgG1 Fc domain; an IgG1hinge; or a CD8 stalk.
 7. A CAR according to claim 1 that comprises anintracellular T-cell signalling domain.
 8. The CAR according to claim 7,wherein the intracellular T-cell signalling domain comprises one or moreof the following endodomains: CD28 endodomain; 41 BB endodomain, OX40endodomain and the CD3-Zeta endodomain.
 9. The CAR according to claim 8,wherein the intracellular T-cell signalling domain comprises: (i) the 41BB endodomain and the CD3-Zeta endodomain; (ii) the OX40 endodomain andthe CD3-Zeta endodomain; or (iii) all of the following endodomains: CD28endodomain; OX40 and CD3-Zeta endodomain.
 10. The CAR according to claim1, which comprises the sequence shown as any of SEQ ID NOs: 10 to 15, ora variant thereof which has at least 80% sequence identity and retainsthe capacity to i) bind CD19 and ii) induce T cell signalling.
 11. TheCAR according to claim 1 which, when expressed by a T-cell and used totarget a CD19-expressing cell, causes lower IFNγ release by theCD19-expressing target cell than that caused by a T-cell expressing aCAR comprising a CD19-binding domain which comprises a) a heavy chainvariable region (VH) having complementarity determining regions (CDRs)with the following sequences: CDR1 (SEQ ID NO. 16) GVSLPDY; CDR2(SEQ ID NO. 17) WGSET; and CDR3 (SEQ ID NO. 18) HYYYGGSYAMDY;

and b) a light chain variable region (VL) having CDRs with the followingsequences: CDR1 (SEQ ID NO. 19) RASQDISKYLN; CDR2 (SEQ ID NO. 20)HTSRLHS; and CDR3 (SEQ ID NO. 21) QQGNTLPYT.


12. The CAR according to claim 1, wherein the CDRs are grafted on to ahuman or humanised framework.
 13. A nucleic acid comprising a nucleicacid sequence which encodes a CAR, wherein the CAR comprises aCD19-binding domain which comprises: a) a heavy chain variable region(VH) having complementarity determining regions (CDRs) with thefollowing sequences: (SEQ ID NO: 1) CDR1 - GYAFSSS; (SEQ ID NO: 2)CDR2 - YPGDED (SEQ ID NO: 3) CDR3 - SLLYGDYLDY;

and b) a light chain variable region (VL) having CDRs with the followingsequences: (SEQ ID NO: 4) CDR1 - SASSSVSYMH; (SEQ ID NO: 5)CDR2 - DTSKLAS; and (SEQ ID NO: 6) CDR3 - QQWNINPLT.


14. The nucleic acid according to claim 13, wherein the CD19 bindingdomain comprises a VH domain having the sequence shown as SEQ ID NO: 7and/or or a VL domain having the sequence shown as SEQ ID NO: 8 or avariant thereof having at least 95% sequence identity.
 15. The nucleicacid according to claim 13, wherein the CD19 binding domain comprisesthe sequence shown as SEQ ID NO: 9 or a variant thereof having at least90% sequence identity.
 16. The nucleic acid according to claim 13,wherein the CAR comprises the sequence shown as any of SEQ ID NOs: 10 to15, or a variant thereof which has at least 80% sequence identity andretains the capacity to i) bind CD19 and ii) induce T cell signalling.17. A vector which comprises a nucleic acid according to claim
 13. 18. Acell which comprises a CAR according to claim
 1. 19. The cell accordingto claim 18 that is a T cell or a natural killer (NK) cell.
 20. A cellcomposition which comprises a plurality of cells according to claim 18.21. A method for making a cell which comprises a CAR, the methodcomprising a step of transducing or transfecting a cell with a vectoraccording to claim
 17. 22. A method for making a composition whichcomprises a plurality of cells which comprise a CAR, the methodcomprising a step of transducing or transfecting a sample of cells froma subject ex vivo with a vector according to claim
 17. 23. Apharmaceutical composition which comprises a cell according to claim 18,or a plurality of said cells, together with a pharmaceuticallyacceptable carrier, diluent, or excipient.
 24. A method of treating a Bcell malignancy, the method comprising administering to a subject withthe B cell malignancy: a cell according to claim 18, or a pharmaceuticalcomposition which comprises said cell or a plurality of said cells,together with a pharmaceutically acceptable carrier, diluent, orexcipient.